The protein interaction map of bacteriophage lambda

<p>Abstract</p> <p>Background</p> <p>Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage.</p> <p>Results</p> <p>In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.</p> <p>Conclusions</p> <p>Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.</p

Transcriptional activators in yeast

Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2)

The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology

<p>Abstract</p> <p>Background</p> <p>Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion.</p> <p>Results</p> <p>We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known <it>Escherichia coli </it>K-12 ORFs in Gateway^®entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in <it>E. coli</it>, <it>Streptococcus pneumoniae</it>, and <it>Staphylococcus aureus</it>. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics.</p> <p>Conclusions</p> <p>The <it>E. coli </it>ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.</p

The Binary Protein Interactome of Treponema pallidum – The Syphilis Spirochete

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

From TgO/GABA-AT, GABA, and T-263 mutant to conception of Toxoplasma

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat’s oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with “Rosetta stone”-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite’s capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease

Evolutionarily Conserved Herpesviral Protein Interaction Networks

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species

Novel Conserved Assembly Factor of the Bacterial Flagellum

TP0658 (FliW) and its orthologs, conserved proteins of unknown function in Treponema pallidum and other species, interact with a C-terminal region of flagellin (FlaB1-3 in T. pallidum; FliC in most other species). Mutants of orthologs in Bacillus subtilis and Campylobacter jejuni (yviF, CJ1075) showed strongly reduced motility. TP0658 stabilizes flagellin in a way similar to FliS, suggesting that TP0658 is a conserved assembly factor for the bacterial flagellum

Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread